A sensitive enzyme assay for biotin, avidin, and streptavidin

Reciprocal enzyme assays are described for the vitamin biotin and for the biotin-binding proteins avidin and streptavidin. The assays are based on the following steps: (a) biotinylated bovine serum albumin is adsorbed onto microtiter plates; (b) streptavidin (or avidin) is bound to the biotin-coated plates; (c) biotinylated enzyme (in this case alkaline phosphatase) is then interacted with the free biotin-binding sites on the immobilized protein. For biotin assay, competition between the free vitamin and the biotinylated enzyme is carried out between steps (b) and (c). The method takes advantage of the four biotin-binding sites which characterize both avidin and streptavidin. The method is extremely versatile and accurate over a concentration range exceeding three orders of magnitude. The lower limits of detection are approximately 2 pg/ml (0.2 pg/sample) for biotin and less than 100 ng/ml (10 ng/sample) for either avidin or streptavidin.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions

We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

生物素化荧光素酶的克隆表达及其固定化研究

为了在体内实现萤火虫荧光素酶的生物素酰化修饰,我们将大肠杆菌中编码生物素羧基载体蛋白（biotin carboxyl carrier protein,BCCP）C端87个氨基酸的功能域基因融合到萤火虫(Pyrocoelia pectoralis)荧光素酶cDNA的末端。经大肠杆菌生物素合成酶（biotin holoenzyme synthetase）的催化,生物素与BCCP上特定的赖氨酸（Lys）残基共价结合,由此与BCCP融合的萤火虫荧光素酶间接实现了生物素化的修饰。利用生物素与配体亲和素或链霉亲和素的特异性耦合,可以将荧光素酶固定到亲和素或链霉亲和素包被的磁珠上,从而使荧光检测的应用更加灵活和方便。本文将就生物素化荧光素酶的克隆、表达以及功能检测进行具体讨论。     

A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase.

A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal beta 1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal beta 1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc alpha 2-6Gal beta 1-4GlcNAc-R. The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in alpha-2,6, but not in alpha-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 microU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 microliters of serum.     

Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.

Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is under control of the lambda PL promoter [Cormier et al. (1989) Photochem. Photobiol. 49, 509]. Purification of recombinant apoaequorin from E. coli containing this new recombinant plasmid (pAEQ1.3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of recombinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubation with synthetic coelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10(-18) and 10(-12) mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 degrees C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotin interaction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.     

A streptavidin mutant useful for directed immobilization on solid surfaces.

A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

Instability of the biotin-protein bond in human plasma

Labeling proteins with biotin offers an alternative to labeling with radioisotopes for pharmacokinetic studies in humans. However, stability of the biotin-protein bond is a critical tacit assumption. Using release of biotin from immunoglobulin G as the outcome, we individually evaluated stability of the biotin label produced by six biotinylation agents: biotin PEO-amine, 5-(biotinamido)-pentylamine, iodoacetyl-LC-biotin, NHS-LC-biotin, sulfo-NHS-LC-biotin, and biotin-LC-hydrazide. Each of the six biotinylated proteins was incubated at room temperature for 4h in human plasma or in phosphate-buffered saline (control). Free biotin was separated from the biotinylated protein by ultrafiltration and quantitated by avidin-binding assay. For each biotinylation reagent, biotin release was significantly increased by plasma (p < 0.0001 vs control by unpaired t test). Moreover, the hydrazide bond was also unstable in buffer. Biotin remaining on the protein was quantitated directly using capture of europium-streptavidin by the immobilized biotinylated immunoglobulin G. Consistent with biotin release data, streptavidin capture was reduced by plasma to 8% of control. We conclude that all of the biotinylating agents produce biotin-protein bonds that are susceptible to hydrolysis by factors present in human plasma; five of six are stable in buffer.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin

This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.     

A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles.

The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.     

Ligands for insulin receptor isolation

Biotinylated insulins are bivalent molecules having the ability to bind to insulin receptors on the one hand and to "avidins" on the other. In order to be useful as ligands for insulin receptor isolation, biotinylated insulins must be developed that have the capacity to bind simultaneously to both and insulin receptor. The present investigation addresses this problem. A series of biotinylated and dethiobiotinylated insulins has been prepared in which the distance between the biotin carboxyl group and the insulin varies from 7 to 20 atoms. These compounds form complexes with succinoylavidin. The dissociation rates (K-1) of these complexes have been determined from the [14C]biotin exchange assay. The dissociation kinetics of most of these complexes are biphasic, and the kinetic constants reported are those corresponding to the slow rate. Ligands containing dethiobiotin dissociate more rapidly than the corresponding biotin derivatives. The interposition of a spacer arm substantially decreases the rate of dissociation. The [14C]biotin exchange assay could not be used with streptavidin complexes of the above ligand since biotin dissociates more rapidly from streptavidin than from succinoylavidin. However, the relative dissociation rates of a series of ligands could be determined and were as follows: 6-(dethiobiotinylamido)-hexanoic acid greater than dethiobiotinyl-A1-insulin greater than biotinylinsulin greater than biotinyl-A1-insulin greater than biotinyl-A2-insulin. Dethiobiotin and its amide failed to form complexes with streptavidin. The affinity of the ligands for insulin receptors was determined by measuring their ability to stimulate 14CO2 formation from [1-14C]glucose in rat epididymal adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.